The invention relates to a method of determining at least a measure of a density of markers in a sample.
Fluorescence microscopy is an important imaging method for biological and bio-medical sciences because functional biological molecules like proteins can be visualized through the attachment of a marker. Such a marker is a small fluorescent molecule that, upon illumination with light of a specific color, emits light of a slightly different color and that can be attached to a protein of interest. By filtering the illumination light, the marked proteins become visible onto a dark background. The marker molecule can be of non-biological origin (i.e. exogenous), in which case it can be linked to biological molecules via a linker molecules such as anti-bodies. This is usually referred to as immuno-labelling. Alternatively, the maker molecule can be genetically expressed, in which case it is already present in the biological material itself, i.e. endogenous. These fluorescent markers mostly belong to the class of fluorescent proteins. In most cases the function of a protein or of a group of proteins is intricately linked to the biological structure onto which the protein is attached. Such structures can be microtubules, fibers, mitochondria, organelles, the nucleus, particles, etc. In a biological cell, these structures typically have one or more dimensions that are below the diffraction limit for optical microscopy, i.e. smaller than 300 nm.
In principle, the position of a fluorescent molecule can be retrieved at much higher accuracy than that imposed by the diffraction limit. Localization accuracy down to about 20 nm has been reported, provided one or only a few fluorescent molecules are contained within an area equal to the size of the diffraction-limited spot. The localization procedure is done through data analysis of the measured optical intensity gradient (de-convolution with the optical Point Spread Function, Gaussian fitting).
In case more than a single molecule is located within the diffraction-limited spot their individual positions can still be retrieved provided that discrete bleaching of each individual molecule can be observed in the recorded optical intensity. Bleaching refers to a change in the fluorescent molecule that makes the molecule non-fluorescent. This bleaching can also be caused by an induced destruction of a fluorescent molecule, e.g. through irradiation by a focused particle beam.
Such a method with induced destruction is for example disclosed in the European Patent Application 1.655.597. This patent application describes a method of determining the position of fluorescent markers in a sample with a high spatial resolution. To this end, the sample is illuminated with a light beam, while the sample is simultaneously scanned by a particle beam. In response to the excitation by the light beam, a flux of fluorescence radiation is emitted from the sample. This flux is generated by one or more markers in the sample that are located in the illuminated region. During scanning, markers will be impinged upon by the particle beam and will be damaged in such a manner that the marker impinged upon will no longer emit fluorescent radiation. This leads to a reduction of the flux of fluorescent radiation. This reduction is detected. If the flux that results from excitation decreases by at least a previously determined threshold value, this has to be because a marker in the excitation region is damaged by the particle beam. Since the position of the particle beam with respect to the sample is known at the moment that the marker is damaged, the position of the marker in the sample is, accordingly, also known.
The same procedure of identifying the location of individual markers by determining damage events which are represented by a vertical drop in the fluorescence signal, is also described in EP 2.482.061. In EP 2.482.061 it is described that this method can also be done in a scan field containing 10000 green fluorescent proteins (GFPs), although the distinguishing of individual GFP damage events from out of the overall statistical noise in the light signal will be more difficult when compared with a sample having less GFPs, for example 100 GFPs, in the scan field.
As the discrete individual bleaching steps have to be observed above the background of the remaining fluorescent molecules, localization is typically limited to marker densities of 15-20 molecules per diffraction area. For higher densities the detection of a signal of a single molecule in view of the noise in the signal from other molecules, is very difficult.
However as proteins involved in a biological reaction assemble at a biological structure with one or more dimensions in the 10-100 nm size range, the local density of fluorescent markers is typically much higher than that allowed for localization techniques that rely on discrete bleaching steps as for example described in European Patent Applications 1.655.597 and 2.482.061. In a structure of interest there are usually so many fluorescent markers that it is impossible to identify the signal from a single marker in the total fluorescence signal within the diffraction spot. In addition, in real life samples, the total numbers of fluorescent makers in a scan field is not known upfront, as is the case in the examples described in EP 2.482.061.
A further disadvantage for using the method as described in EP 2.482.061 is, that every marker in the scan field is destroyed by scanning the charged particle beam over the same area as irradiated by the laser beam. The part of the sample which has been scanned by the charged particle beam is therefore permanently damaged and cannot be used for further fluorescence measurements.
It is the aim of the present invention to provide an alternative and novel technique for studying fluorescent markers in a sample.